Polymerase Chain Reaction (PCR) is a DNA amplification technique which is essential to genetics, and particularly in next generation sequencing, where amplification of the quantity of starting DNA is commonly performed. It is an example where technology and basic research have been combined to deliver a tool that has been applied to a multitude of fields such as genomics, forensics, DNA/RNA aptamers optimization, and diagnostic testing.
PCR is a temperature mediated process that requires cycling between set temperatures. Single strand DNA is required for two primer sequences to bind upstream and downstream of the region to be amplified. To allow this to occur, the first step is denaturation or separation of the two strands at around 94-98° C. Primer annealing occurs around 45-55° C. and allows the thermo-stable polymerase to bind to defined regions of double stranded DNA. The next stage is elongation of the double stranded copy where the temperature is raised to the optimum temperature (around 72° C.) for the enzyme catalysis to proceed. Finally, temperature is returned to 94° C. for denaturation to single stranded DNA that allows the cycle repeat.
The thermal cycler (also known as a Thermocycler, PCR Machine or DNA Amplifier) is an apparatus used to amplify segments of DNA via the polymerase chain reaction (PCR) process. Thermal cyclers are typically provided with a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted. Heat is provided through solid state heaters or infrared lamps. The cycler raises and lowers the temperature of the thermal block in discrete, preprogrammed steps.
There is a need if the field to increase the speed, and therefore the efficiency, of PCR processes. The duration of the thermocycling of a PCR process can be dependent upon several factors, including the experimentalist's requirements. Indeed, for a molecular biologist involved in sequencing large sections of a genome, amplification of large fragments would require a longer cycle time than in for more commonplace diagnostic applications, for example, to ensure high yield. The additional time required is a function of the temperature ramp time and cooling between the stages of PCR (Denaturation, primer annealing and elongation/synthesis). Shortening ramp and cooling times means more rapid transition and shorter cycling times, even appreciating for long fragments, a more substantial pause at the elongation temperature is required reflecting the polymerisation rate of the enzyme, expressed in base pairs per second (ranging from a few hundred to 1 kilobase per second). The cycle time can be shortened with more rapid enzymes or by allowing incomplete amplification of amplicons that are termed mega-primers to be completed in subsequent cycles. Though the later technique lowers the overall yield from 30 cycles it does allow slower polymerases to be utilised. More fundamental is that very few instruments on the market are available of delivering cycle times of less than 7 minutes, to full exploit rapid cycling and at a cost suitable to wide spread application.
An example of such thermocycler is the Lightcycler® that has been commercialized by Roche. The Lightcycler® can achieve heating rates of 15° C. per second with cooling rates of 10° C. per second, but commonly ramp times are significantly less than this, at around 2-5° C. per second (heating), reflecting heat delivery by Peltier elements that struggle to produce rapid heating of aluminium or ceramic blocks used to hold tubes.
Another downside of commercial real time quantitative thermal cyclers known in the art is the cost of each instrument, running into tens of thousands of dollars for rapid thermocycling. The current high cost of all PCR thermocycler platforms (real time PCR inclusive) represents a significant research cost to the experimentalist. PCR is the backbone of many molecular biological studies since its popularization by Nobel Laureate Kary Mullis and improvements to both method and instrument are always sought.
The biological components have been demonstrated to be able to run much faster than common instrumental cycle times. It would therefore be advantageous to provide an instrument which scales and lowers the cost burden such that its use becomes more widespread, while still delivering sub 10 minute reaction times for 30 cycles.
Other DNA amplifications are known in the art. One example is Loop-mediated isothermal amplification (LAMP), which involves holding a temperature (for example 65° C.) to allow Bst enzymes to perform a loop amplification using specially designed primers, to cause the formation of one massive repeating chain DNA extended polymer. Although ramping and cycling times are less of an issue, it is still desirable to provide an efficient and economical means to control the temperature of the reaction. The same can be said for any DNA amplification technique where heat needs to be applied to the reaction mixture.
Heating a reaction mixture that contains a DNA molecule is not only useful for DNA extraction techniques but is also used for other DNA-involving processes, such as cell lysis and sample sterilization.
There is therefore a need for a heating method and device for reaction mixtures containing DNA which alleviates at least some of the aforementioned drawbacks.